Human Immunodeficiency Virus-1 Tat Induces Hyperproliferation and Dysregulation of Renal Glomerular Epithelial Cells
2002; Elsevier BV; Volume: 161; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64156-9
ISSN1525-2191
AutoresPier Giulio Conaldi, Antonella Bottelli, Andreina Baj, Caterina Serra, Lisa B. Fiore, Giovanni Federico, Benedetta Bussolati, Giovanni Camussi,
Tópico(s)HIV/AIDS drug development and treatment
ResumoHuman immunodeficiency virus-associated nephropathy (HIVAN) is etiologically related to the viral infection, but the mechanisms of virus-induced renal injury remain undetermined. Peculiar histopathological features of HIVAN are the enhanced proliferation and the loss of differentiation markers of glomerular epithelial cells (podocytes). We found that podocytes were not permissive to HIV-1 replication. In this study we investigated the effects of the HIV-1 regulatory protein Tat on primary cultures and on a continuous line of podocytes. Our results demonstrated that Tat induced hyperproliferation of these cells in a dose-dependent manner. This activity was primarily mediated by the basic domain of the viral protein. Proteoglycans were required for this phenomenon because Tat-induced increase of podocyte growth was significantly impaired by inhibition of proteoglycan synthesis with β-d-xyloside. In podocyte cultures Tat promoted both the transcription and the release of basic fibroblast growth factor, which contributed to the enhanced cell proliferation. Moreover, Tat deregulated the podocyte phenotype causing down-regulation of maturity markers such as WT-1 and synaptopodin, alteration of cytoarchitecture, and impairment of permselectivity. Together, these results demonstrate that the interaction of extracellular Tat with podocytes can induce alterations that mimic the pathological changes of podocytes detected in HIVAN. Human immunodeficiency virus-associated nephropathy (HIVAN) is etiologically related to the viral infection, but the mechanisms of virus-induced renal injury remain undetermined. Peculiar histopathological features of HIVAN are the enhanced proliferation and the loss of differentiation markers of glomerular epithelial cells (podocytes). We found that podocytes were not permissive to HIV-1 replication. In this study we investigated the effects of the HIV-1 regulatory protein Tat on primary cultures and on a continuous line of podocytes. Our results demonstrated that Tat induced hyperproliferation of these cells in a dose-dependent manner. This activity was primarily mediated by the basic domain of the viral protein. Proteoglycans were required for this phenomenon because Tat-induced increase of podocyte growth was significantly impaired by inhibition of proteoglycan synthesis with β-d-xyloside. In podocyte cultures Tat promoted both the transcription and the release of basic fibroblast growth factor, which contributed to the enhanced cell proliferation. Moreover, Tat deregulated the podocyte phenotype causing down-regulation of maturity markers such as WT-1 and synaptopodin, alteration of cytoarchitecture, and impairment of permselectivity. Together, these results demonstrate that the interaction of extracellular Tat with podocytes can induce alterations that mimic the pathological changes of podocytes detected in HIVAN. A relevant increase in kidney disease incidence has been recently reported in human immunodeficiency virus (HIV)-infected patients.1Winston JA Burns GC Klotman PE The human immunodeficiency virus (HIV) epidemic and HIV-associated nephropathy.Semin Nephrol. 1998; 18: 373-377PubMed Google Scholar HIV-associated nephropathy (HIVAN) is the prominent renal complication of HIV infection, characterized by heavy proteinuria and progressive renal failure with severe prognosis.2D'Agati V Appel GB Renal pathology of human immunodeficiency virus infection.Semin Nephrol. 1998; 18: 406-421PubMed Google Scholar, 3Klotman PE HIV-associated nephropathy.Kidney Int. 1999; 56: 1161-1176Crossref PubMed Scopus (91) Google Scholar If untreated, HIVAN may progress to end-stage renal disease in few months. In African-American adults, HIVAN is the third leading cause of end-stage renal disease.1Winston JA Burns GC Klotman PE The human immunodeficiency virus (HIV) epidemic and HIV-associated nephropathy.Semin Nephrol. 1998; 18: 373-377PubMed Google Scholar HIVAN is considered a pan-nephropathy2D'Agati V Appel GB Renal pathology of human immunodeficiency virus infection.Semin Nephrol. 1998; 18: 406-421PubMed Google Scholar characterized, on the pathological point of view, by a collapsing form of glomerulosclerosis associated with severe tubulointerstitial injury (microcystic dilatation and apoptotic changes of tubuli and mononuclear infiltration). It has been recently shown that glomerular lesions of HIVAN involve striking alterations of glomerular epithelial cells (podocytes), which include hyperplasia and dysregulation of mature podocyte phenotypic markers.4Barisoni L Kriz W Mundel P D'Agati V The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy.J Am Soc Nephrol. 1999; 10: 51-61Crossref PubMed Scopus (5) Google Scholar Clinical and experimental data indicate that HIVAN is etiologically related to HIV infection, but the pathogenesis of virus-induced renal injury is not clearly understood.5Schwartz EJ Klotman PE Pathogenesis of human immunodeficiency virus (HIV)-associated nephropathy.Semin Nephrol. 1998; 18: 436-445PubMed Google Scholar We recently reported that tubular epithelial cells and glomerular mesangial cells (MCs) could sustain viral productive replication with distinct pathological effects that mimic the alterations detected in HIVAN. In cultured tubular epithelial cells, HIV-1 caused apoptosis,6Conaldi PG Biancone L Bottelli A Wade-Evans A Racusen LC Boccellino M Orlandi V Serra C Camussi G Toniolo A HIV-1 kills renal tubular epithelial cells in vitro by triggering an apoptotic pathway involving caspase activation and Fas upregulation.J Clin Invest. 1998; 102: 2041-2049Crossref PubMed Scopus (145) Google Scholar whereas in MCs HIV-1 established persistent infection and stimulated the synthesis of cytokines involved in the pathogenesis of glomerulosclerosis.7Conaldi PG Bottelli A Wade-Evans A Biancone L Baj A Cataluppi V Serra C Dolei A Toniolo A Camussi G HIV-persistent infection and cytokine induction in mesangial cells: a potential mechanism for HIV-associated glomerulosclerosis.AIDS. 2000; 14: 2045-2047Crossref PubMed Scopus (22) Google Scholar Conversely, podocytes were not permissive to virus replication, possibly because they did not express HIV-1 receptors.7Conaldi PG Bottelli A Wade-Evans A Biancone L Baj A Cataluppi V Serra C Dolei A Toniolo A Camussi G HIV-persistent infection and cytokine induction in mesangial cells: a potential mechanism for HIV-associated glomerulosclerosis.AIDS. 2000; 14: 2045-2047Crossref PubMed Scopus (22) Google Scholar A HIVAN-like disease characterized by podocyte dysregulation occurred in transgenic mice with intrarenal expression of gag-pol-deleted HIV provirus. In these transgenic mice the synthesis of unspliced and multiply spliced viral regulatory proteins (Tat, Rev, and Nef) occurred in renal cells in the absence of a productive viral replication.8Bruggeman LA Dikman S Meng C Quaggin SE Coffman TM Klotman PE Nephropathy in human immunodeficiency virus-1 transgenic mice is due to renal transgene expression.J Clin Invest. 1997; 100: 84-92Crossref PubMed Scopus (229) Google Scholar, 9Barisoni L Bruggeman LA Mundel P D'Agati VD Klotman PE HIV-1 induces renal epithelial dedifferentiation in a transgenic model of HIV-associated nephropathy.Kidney Int. 2000; 58: 173-181Crossref PubMed Scopus (169) Google Scholar Thus, podocyte alterations could be caused by the expression of these viral proteins. In the present study, we investigated the effects of Tat on human cultured podocytes. HIV-1 Tat is a transactivating factor of 86 to 101 amino acids, which is encoded by two exons and is translated from multiply spliced 2-kb mRNAs.10Vaishnav YN Wong-Staal F The biochemistry of AIDS.Annu Rev Biochem. 1991; 60: 577-630Crossref PubMed Scopus (280) Google Scholar Tat is essential for HIV-1 replication and it is released by HIV-infected cells, as demonstrated by the detection of Tat in the sera of HIV patients.11Ensoli B Buonaguro L Barillari G Fiorelli V Gendelman R Morgan RA Wingfield P Gallo RC Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.J Virol. 1993; 67: 277-287Crossref PubMed Google Scholar, 12Westendorp MO Frank R Ochsenbauer C Striker K Dhein J Walczak H Debatin KM Krammer PH Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.Nature. 1995; 375: 497-500Crossref PubMed Scopus (912) Google Scholar Extracellular Tat exerts pleiotropic effects on uninfected cells either entering into the cells or interacting with cell-surface receptors. In fact, Tat has been shown to stimulate proliferation, migration, and production of growth factors and cytokines in several cell types.13Barillari G Gendelman R Gallo RC Ensoli B The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence.Proc Natl Acad Sci USA. 1993; 90: 7941-7945Crossref PubMed Scopus (340) Google Scholar, 14Lotz M Clark-Lewis I Ganu V HIV-1 transactivator protein Tat induces proliferation and TGF beta expression in articular chondrocytes.J Cell Biol. 1994; 124: 365-371Crossref PubMed Scopus (77) Google Scholar, 15Barillari G Sgadari C Fiorelli V Samaniego F Colombini S Manzari V Modesti A Nair BC Cafaro A Sturzl M Ensoli B The Tat protein of human immunodeficiency virus type-1 promotes vascular cell growth and locomotion by engaging the α5β1 and αvβ3 integrins and by mobilizing sequestered basic fibroblast growth factor.Blood. 1999; 94: 663-672PubMed Google Scholar, 16Del Sorbo L De Martino A Biancone L Conaldi PG Toniolo A Camussi G The synthesis of platelet-activating factor mediates chemotaxis of monocytes induced by HIV-1 Tat.Eur J Immunol. 1999; 29: 1513-1521Crossref PubMed Scopus (19) Google Scholar Tat can also elicit apoptotic effects on T cells and neurons12Westendorp MO Frank R Ochsenbauer C Striker K Dhein J Walczak H Debatin KM Krammer PH Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.Nature. 1995; 375: 497-500Crossref PubMed Scopus (912) Google Scholar, 17Li CJ Friedman DJ Wang C Metelev V Pardee AB Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein.Science. 1995; 268: 429-431Crossref PubMed Scopus (546) Google Scholar, 18Nath A Psooy K Martin C Knudsen B Magnuson DS Haughey N Geiger JD Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic.J Virol. 1996; 70: 1475-1480Crossref PubMed Google Scholar whereas it may induce cell survival in other cell types such as endothelial and Kaposi's sarcoma cells.19Cantaluppi V Biancone L Boccellino M Doublier S Benelli R Carlone S Albini A Camussi G HIV type 1 Tat protein is a survival factor for Kaposi's sarcoma and endothelial cells.AIDS Res Hum Retroviruses. 2001; 17: 965-976Crossref PubMed Scopus (35) Google Scholar Remarkably, Tat can induce opposite effects (promotion versus inhibition) on cell survival on the same cell type, depending on its concentration.11Ensoli B Buonaguro L Barillari G Fiorelli V Gendelman R Morgan RA Wingfield P Gallo RC Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.J Virol. 1993; 67: 277-287Crossref PubMed Google Scholar Thus, there is an increasing body of evidence for the role of extracellular Tat in AIDS-associated immune dysfunction and pathological manifestations.20Cohen SS Li C Ding L Cao Y Pardee AB Shevach EM Cohen DI Pronounced acute immunosuppression in vivo mediated by HIV Tat challenge.Proc Natl Acad Sci USA. 1999; 96: 10842-10847Crossref PubMed Scopus (88) Google Scholar Herein we report that HIV-1 Tat affects the podocyte phenotype and functions inducing cell proliferation, down-regulation of mature phenotypic markers, and alteration of cytoarchitecture. Reagents were obtained from the following sources: HIV-1 Tat, rabbit anti-Tat serum from Intracel (London, UK); [11-24], [46-60] Tat peptides from Neosystem (Strasbourg, France); [61-86] Tat peptide from Tecnogen (Piana di Monte Verna, Italy); monoclonal antibody (mAb) against Ki-67 antigen, biotinylated anti-mouse IgG, biotinylated anti-rabbit IgG, and peroxidase-conjugated streptavidin from DAKO (Glostrup, Denmark); mAb anti-synaptopodin, (kindlyprovided by Dr. P. Mundel, Division of Nephrology, Albert Einstein College of Medicine, Bronx, NY, USA); anti-Wilms' tumor antigen (WT-1) rabbit polyclonal Ab, anti-Flk-1 rabbit polyclonal Ab, and anti-Flt-1 rabbit polyclonal Ab from Santa Cruz Biotechnology (Santa Cruz, CA); mAb anti-basic fibroblast growth factor (bFGF) from R&D (Minneapolis, MN); mAb anti-human integrin α3β1, mAb anti-human integrin αvβ3, and mAb anti-human integrin β1 from Chemicon Int. (Temecula, CA); fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse and anti-rabbit IgG, FITC-labeled phalloidin, β-d-xyloside, (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxy-anilide inner salt) sodium salt (XTT), and other immune reagents and chemicals were from Sigma Chemical Co. (St. Louis, MO), unless otherwise specified. Tissue culture media were from Euroclone (Paignton, UK) and fetal calf serum (FCS) from Hyclone (Logan, UT); molecular biology reagents were from Perkin-Elmer Cetus (Norwalk, CT); plastic-ware from Falcon (Becton-Dickinson, San Jose, CA). Primary cultures of glomerular mesangial and epithelial cells were established as described.21Conaldi PG Biancone L Bottelli A De Martino A Camussi G Toniolo A Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.J Virol. 1997; 71: 9180-9187PubMed Google Scholar Briefly, decapsulated glomeruli were isolated by differential sieving from renal cortex fragments taken from surgically removed kidneys of six Caucasian patients, treated with collagenase and seeded in Dulbecco's modified Eagle's medium (DMEM) containing 20% FCS and d-valine. After 2 weeks MCs were collected by digestion with trypsin-ethylenediaminetetraacetic acid and grown in DMEM-10% FCS. Cultured cells were characterized according to established criteria.21Conaldi PG Biancone L Bottelli A De Martino A Camussi G Toniolo A Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.J Virol. 1997; 71: 9180-9187PubMed Google Scholar, 22Striker GE Striker LJ Glomerular cell culture.Lab Invest. 1985; 53: 122-131PubMed Google Scholar, 23Delarue F Virone A Hagege J Lacave R Peraldi M-N Adida C Rondeau E Feunteun J Sraer J-D Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells.Kidney Int. 1991; 40: 906-912Crossref PubMed Scopus (77) Google Scholar Primary cultures of podocytes were obtained by plating at high-density glomeruli untreated with collagenase. After a 10 day-incubation in DMEM-20% FCS, the cultures were trypsinized and the outgrowing podocytes were expanded. Phenotypic characterization was performed according to cell morphology (polyhedral cells with cobblestone-like appearance); positive staining for synaptopodin, WT-1, cytokeratins, vimentin, and laminin; negative staining for smooth muscle-type myosin, FVIIIr:Ag, and CD45; and cytotoxicity in response to 50 μg/ml of puromycin aminonucleoside. Established lines of differentiated MCs and podocytes were obtained by infection of pure primary cultures with a hybrid Adeno5/SV40 virus, as described.21Conaldi PG Biancone L Bottelli A De Martino A Camussi G Toniolo A Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.J Virol. 1997; 71: 9180-9187PubMed Google Scholar Individual foci of immortalized cells were subcultured and cloned. The selected clones were used between passages 30 and 45. The podocyte line was characterized according to the phenotype, following the criteria mentioned above. MCs and podocytes of primary cultures and continuous lines were seeded at the concentration of 2 × 104 cells/cm2 and rested for 5 hours in DMEM-2% FCS. Then, the cells were washed and stimulated with different doses of Tat (0.2 to 200 ng/ml) in medium containing 0.1% bovine serum albumin (BSA). To evaluate the kinetics of the cell growth, the cultures were incubated in medium without phenol red containing 200 μg/ml of XTT and 0.2% phenazine methosulphate and the absorbance at 450 nm was monitored at different times.24Roehm NW Rodgers GH Hatfield SM Glasebrook AL An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.J Immunol Methods. 1991; 142: 257-265Crossref PubMed Scopus (858) Google Scholar The results were confirmed by viable cell counting with the Trypan blue exclusion test. Podocyte proliferation was also measured by the [3H]-thymidine uptake method. Each culture condition was tested in triplicate incubating the cells for 48 hours in 96-well plates as described. During the last 6 hours 1 μCi/well of [3H]-thymidine (Amersham Pharmacia Biotech, Little Chalfont, UK) was added to the cultures. The harvested samples were processed for β-scintillation counting. Experiments were performed by stimulating podocytes with Tat peptides at concentrations equimolar to those of Tat protein. Proliferation tests were also performed by plating podocytes into 96-well plates coated with Tat [1 μg/ml in phosphate-buffered saline (PBS), pH 7.4, for 12 hours at 4°C]. After washing the plates with PBS, the podocytes were seeded at the usual concentration and their growth was evaluated as described. Blocking experiments were done by preincubating podocyte cultures with antibodies (Abs) specific to surface molecules (5 μg/ml in DMEM-0.1% BSA for 1.5 hours at 37°C). To inhibit the synthesis of proteoglycans, β-d-xyloside (25 mmol/L) was added to podocyte cultures 24 hours before stimulation with Tat or Tat peptides. To evaluate the influence of soluble factors in Tat-induced hyperproliferation, podocyte cultures were stimulated with 10 ng/ml of Tat for 1 hour at 37°C or incubated with medium alone, washed for three times and then incubated with DMEM-0.1% BSA. After a 24-hour incubation the supernatants of Tat- and mock-stimulated cultures were taken and centrifuged at 15,000 rpm for 15 minutes at 4°C, then the media were absorbed overnight at 4°C into microtiter plates coated with anti-bFGF mAb (10 μg/ml in PBS pH 7.4 for 12 hours at 4°C) or with anti-Ki67 mAb, used as control antibody. Podocyte cultures were incubated for 24 hours at 37°C with 50% DMEM-0.1%BSA and 50% conditioned media. Cell proliferation was evaluated by viable cell counting and the XTT assay. Podocytes were cultured in chamber slides and stimulated with 10 ng/ml of Tat. After a 24-hour incubation the cells were fixed with cold methanol for 15 minutes and washed with PBS. The monolayers were incubated with 1 μg/ml of anti-Ki-67 mAb for 1 hour at room temperature, washed in PBS, stained with FITC-conjugated goat anti-mouse IgG, and examined with an epifluorescence microscope. Control experiments included incubation of the cells with irrelevant mouse isotypic Igs or omission of primary Abs followed by the appropriate labeled secondary Abs. Immunoperoxidase tests were performed on podocyte cultures fixed in 3.5% paraformaldehyde for 15 minutes at room temperature. The monolayers were incubated with 3% hydrogen peroxide to quench endogenous peroxide activity and with PBS-1% BSA to prevent aspecific reactions. Then, the specimens were incubated with primary Abs (2 μg/ml of anti-WT-1 Igs; 1:10 diluted supernatant of hybridoma specific to synaptopodin; 2 μg/ml of irrelevant mouse and rabbit isotypic Igs) for 1 hour at room temperature After washing with Tris-buffered saline, pH 7.4, and 2% Triton X-100, the cells were incubated at room temperature for 10 minutes with biotinylated anti-mouse or anti-rabbit Igs and then for 10 minutes with streptavidin conjugated to horseradish peroxidase. The specimens were stained with 0.03% benzidine and counterstained with hematoxylin and eosin. The expression of integrins (α3β1, αvβ3, β1) and vascular endothelial growth factor (VEGF)-receptors (VEGFRs: Flk-1, Flt-1) in podocytes was evaluated by flow cytometry. Briefly, 106 cells were incubated for 30 minutes at room temperature with 1 μg of the specific Ab and then with FITC-labeled goat anti-mouse or anti-rabbit IgG. After washings with PBS, the cells were analyzed (FACScan, Becton Dickinson). Cytoskeleton alterations were studied on fixed podocytes after permeabilization with 0.1% Triton X-100 in PBS and stained for F-actin with 2 μg/ml of FITC-conjugated phalloidin. The modification of podocyte shape was monitored as described.25Biancone L Cantaluppi V Boccellino M Bussolati B Del Sorbo L Conaldi PG Albini A Toniolo A Camussi G Motility induced by human immunodeficiency virus-1 Tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis.Am J Pathol. 1999; 155: 1731-1739Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar The cells were plated in a hermetically sealed Plexiglas Nikon NP-2 incubator (Nikon Co., Tokyo, Japan) at 37°C, rested for 5 hours with medium containing 2% FCS, stimulated with Tat (1 to 10 ng/ml) and then observed throughout a 2-hour period by Nikon Diaphot inverted microscope with a ×10 phase-contrast objective. The imagines were recorded by a JVC-1CCD video camera (video card Targa 2000; Truevision, Santa Clara, CA) and evaluated by MicroImage-analysis system (Casti Imaging, Venice, Italy). Podocytes were seeded on polycarbonate filters (pore size, 0.4 μm) of Transwell chambers (Costar, Cambridge, MA) at the concentration of 1.5 × 105/well in DMEM-2% FCS. The permeability of the monolayers was measured by diffusion of Trypan blue-albumin complexes.26Camussi G Turello E Bussolino F Baglioni C Tumor necrosis factor alters cytoskeletal organization and barrier function of endothelial cells.Int Arch Allergy Appl Immunol. 1991; 96: 84-91Crossref PubMed Scopus (48) Google Scholar After overnight incubation, 0.2 ml of medium without phenol red and FCS were added in the upper chamber and the cells were stimulated for 1 hour at 37°C with 10 ng/ml of Tat. Then, the upper chamber was filled with 0.3 ml of DMEM-200 mmol/L Trypan blue-albumin. The chambers were incubated at 37°C with continuous agitation. After 5 minutes the monolayers showing leakage of dye were discarded. Transport of albumin across monolayers was determined by sampling aliquots at different times (15 to 120 minutes) and measuring absorbance at 590 nm in the outer and inner wells in duplicate.26Camussi G Turello E Bussolino F Baglioni C Tumor necrosis factor alters cytoskeletal organization and barrier function of endothelial cells.Int Arch Allergy Appl Immunol. 1991; 96: 84-91Crossref PubMed Scopus (48) Google Scholar The expression of bFGF was assessed by reverse transcriptase-polymerase chain reaction (PCR) using total RNA taken from untreated or Tat-stimulated podocytes. cDNA was synthesized by priming RNA (1, 0.1, or 0.01 μg) with oligo-dT (Clontech). PCR was performed using 30 pmol/L of mRNA-specific primers (forward 5′GGCTTCTTCCTGCGCATCC3′; reverse, 5′CCGTACTTTGCTTGACCCG3′) synthesized by Genset (Paris, France).27Jones SG Morrisey K Williams JD Phillips AO TGF-β1 stimulates the release of pre-formed bFGF from renal proximal tubular cells.Kidney Int. 1999; 56: 83-91Crossref PubMed Scopus (26) Google Scholar PCR was performed for only 20 cycles (30 seconds at 94°C, 30 seconds at 57°C, and 30 seconds at 72°C) to detect amplicons of bFGF transcripts (283 bp) under conditions of linear amplification.21Conaldi PG Biancone L Bottelli A De Martino A Camussi G Toniolo A Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.J Virol. 1997; 71: 9180-9187PubMed Google Scholar The production of bFGF by podocytes was evaluated by measuring the bFGF content of podocyte culture supernatants, taken in different experimental conditions, by a commercial enzyme-linked immunosorbent assay kit following the manufacturer's recommendations (R&D Systems, Minneapolis, MN). All data are given as mean values ± SD. Differences among multiple groups were evaluated by one-way analysis of variance in combination with Tukey's multiple comparison test. A P value of <0.05 was considered significant. The treatment of podocytes with exogenous Tat induced a relevant increase of cell proliferation. Figure 1 shows the time-dependent increase in cell proliferation of a continuous line of podocytes after stimulation with Tat at 2 ng/ml. This Tat concentration is comparable to that detected in sera of AIDS patients.12Westendorp MO Frank R Ochsenbauer C Striker K Dhein J Walczak H Debatin KM Krammer PH Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.Nature. 1995; 375: 497-500Crossref PubMed Scopus (912) Google Scholar Tat also stimulated the growth of MCs, but to a lower extent. Similar results were obtained with primary cultures of podocytes and MCs derived from six different donors (data not shown). The proliferative effect of Tat was also detected by [3H]-thymidine uptake assay (data not shown). The Tat-induced proliferation was dose-dependent and occurred at a concentration of Tat as low as 0.2 ng/ml (Figure 2). Heat-treated or neutralized Tat was inactive, demonstrating the specificity of the observed phenomenon. The neutralization of Tat was performed by preincubation with the specific Ab (5 μg/ml for 1 hour at 37°C): the addition of Ab alone did not affect podocyte growth (not shown). By detection of Ki-67 antigen, a nuclear protein expressed by all proliferating cells, a striking increase of the number of cycling cells was observed after the treatment with Tat (vehicle alone, 15 ± 7%; Tat, 90 ± 5%; Figure 3). To elucidate the mechanisms of Tat activity, we tried to identify the protein domains required for this phenomenon. Cell growth experiments were performed using the basic [46-60] peptide and the [61-86] peptide that encompasses the RGD region of Tat. The [11-24] peptide considered biologically unrelevant was used as control.28Jeang K-T Xiao H Rich EA Multifaceted activities of the HIV-1 transactivator of transcription, Tat.J Biol Chem. 1999; 274: 28837-28840Crossref PubMed Scopus (377) Google Scholar As shown in Figure 4, the results indicated that the basic domain of Tat exerted the prominent effect in enhancing podocyte proliferation, but also the RGD region contributed to the activity of the whole protein, as shown by the additive effect obtained by incubating cells with both [46-60] and [61-86] peptides. In contrast, the [11-24] peptide was ineffective on cell proliferation.Figure 2Dose-dependence of the increase of podocyte growth induced by Tat. The results are expressed as the percentage of cell proliferation in comparison with that of untreated cell cultures. Cell growth evaluation was performed after a 48-hour incubation with the XTT assay in five independent experiments, each in triplicate. Similar results were obtained by viable cell counting (100% increase = increase from 3 × 104 cells/cm2 to 5.5 × 104 cells/cm2, on average).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Expression of the proliferation marker Ki-67 in podocytes after a 24-hour incubation. In cultures of untreated podocytes (top) the average number of Ki-67-positive cell cycle-engaged cells was 15 ± 7% of the cells incubated in DMEM-0.1% BSA. After treatment with 10 ng/ml of Tat (bottom), 90 ± 5% of the cultured podocytes expressed the Ki-67 nuclear antigen. Original magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Evaluation of the involvement of different Tat domains in podocyte hyperproliferation. The experiments were performed by using Tat (10 ng/ml) or equimolar concentrations of Tat peptides: [11-24] Tat and [46-60] Tat including the basic domain [61-86] Tat encompassing the RGD region. The data (from five experiments, each in triplicate) refer to the percentage of cell growth after a 48-hour incubation in comparison with that of untreated cells. Podocyte growth was evaluated by the XTT assay (by viable cell counting, 100% increase = increase from 4 × 104 cells/cm2 to 7.5 × 104 cells/cm2, on average).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate whether Tat stimulated podocytes as a result of surface receptor interaction or as a consequence of its internalization, podocyte proliferation was studied by seeding the cells on 96-well plates coated with Tat. After a 48-hour incubation, immobilized Tat stimulated cell proliferation in a way comparable to that of soluble Tat (immobilized Tat, 265 ± 35% increase; 10 ng/ml Tat, 290 ± 30% increase). With other cell types Tat has been shown to modulate cell functions by engaging integrins or VEGFRs via its RGD and basic regions, respectively.13Barillari G Gendelman R Gallo RC Ensoli B The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence.Proc Natl Acad Sci USA. 1993; 90: 7941-7945Crossref PubMed Scopus (
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